Botrytis cinerea in vivo Inoculation Assays for Early-, Middle- and Late-stage Strawberries.

Strawberries are delicious and nutritious fruits that are widely cultivated and consumed around the world, either fresh or in various products such as jam, juice, and ice cream. Botrytis cinerea is a fungal pathogen that causes gray mold disease on many crops, including strawberries. Disease monitoring is an important aspect for growing commercial crops like strawberry because there is an urgent need to develop effective strategies to control this destructive gray mold disease. In this protocol, we provide an important tool to monitor the gray mold fungal infection progression in different developmental stages of strawberry. There are different types of inoculation assays for B. cinerea on strawberry plants, such as in vitro (in/on a culture medium) or in vivo (in a living plant). In vivo inoculation assays can be performed at early, middle, and late stages of strawberry development. Here, we describe three methods for in vivo inoculation assays of B. cinerea on strawberry plants. For early-stage strawberry plants, we modified the traditional fungal disc inoculation method to apply to fungal infection on strawberry leaves. For middle-stage strawberry plants, we developed the flower infection assay by dropping fungal conidia onto flowers. For late-stage strawberry plants, we tracked the survival rate of strawberry fruits after fungal conidia infection. This protocol has been successfully used in both lab and greenhouse conditions. It can be applied to other flowering plants or non-model species with appropriate modifications. Key features • Fungal disc inoculation on early-stage strawberry leaves. • Fungal conidia inoculation on middle-stage strawberry flowers. • Disease rating for late-stage strawberry fruits. • This protocol is applicable to the other flowering plants with appropriate modifications.

Reduced viscosity mutants of Trichoderma reesei with improved industrial fermentation characteristics.

Morphological mutants of Trichoderma reesei were isolated following chemical or insertional mutagenesis. The mutant strains were shown to have reduced viscosity under industrially relevant fermentation conditions and to have maintained high specific productivity of secreted protein. This allowed higher biomass concentration to be maintained during the production phase and, consequently, increased volumetric productivity of secreted protein. The causative mutations were traced to four individual genes (designated sfb3, ssb7, seb1, and mpg1). We showed that two of the morphological mutations could be combined in a single strain to further reduce viscosity and enable a 100% increase in volumetric productivity.

The Scaffold Proteins Paxillin B and α-Actinin Regulate Septation in Aspergillus nidulans via Control of Actin Ring Contraction.

Cytokinesis, as the final step of cell division, plays an important role in fungal growth and proliferation. In the filamentous fungus Aspergillus nidulans, defective cytokinesis is able to induce abnormal multinuclear or nonnucleated cells and then result in reduced hyphal growth and abolished sporulation. Previous studies have reported that a conserved contractile actin ring (CAR) protein complex and the septation initiation network (SIN) signaling kinase cascade are required for cytokinesis and septation; however, little is known about the role(s) of scaffold proteins involved in these two important cellular processes. In this study, we show that a septum-localized scaffold protein paxillin B (PaxB) is essential for cytokinesis/septation in A. nidulans The septation defects observed in a paxB deletion strain resemble those caused by the absence of another identified scaffold protein, α-actinin (AcnA). Deletion of α-actinin (AcnA) leads to undetectable PaxB at the septation site, whereas deletion of paxB does not affect the localization of α-actinin at septa. However, deletion of either α-actinin (acnA) or paxB causes the actin ring to disappear at septation sites during cytokinesis. Notably, overexpression of α-actinin acnA partially rescues the septum defects of the paxB mutant but not vice versa, suggesting AcnA may play a dominant role over that of PaxB for cytokinesis and septation. In addition, PaxB and α-actinin affect the septal dynamic localization of MobA, a conserved component of the SIN pathway, suggesting they may affect the SIN protein complex function at septa. Protein pull-down assays combined with liquid chromatography-mass spectrometry identification indicate that α-actinin AcnA and PaxB likely do not directly interact, but presumably belong to an actin cytoskeleton protein network that is required for the assembly and contraction of the CAR. Taken together, findings in this study provide novel insights into the roles of conserved scaffold proteins during fungal septation in A. nidulans.

Regulation of hyphal morphogenesis by cdc42 and rac1 homologues in Aspergillus nidulans.

The ability of filamentous fungi to form hyphae requires the establishment and maintenance of a stable polarity axis. Based on studies in yeasts and animals, the GTPases Cdc42 and Rac1 are presumed to play a central role in organizing the morphogenetic machinery to enable axis formation and stabilization. Here, we report that Cdc42 (ModA) and Rac1 (RacA) share an overlapping function required for polarity establishment in Aspergillus nidulans. Nevertheless, Cdc42 appears to have a more important role in hyphal morphogenesis in that it alone is required for the timely formation of lateral branches. In addition, we provide genetic evidence suggesting that the polarisome components SepA and SpaA function downstream of Cdc42 in a pathway that may regulate microfilament formation. Finally, we show that microtubules become essential for the establishment of hyphal polarity when the function of either Cdc42 or SepA is compromised. Our results are consistent with the action of parallel Cdc42 and microtubule-based pathways in regulating the formation of a stable axis of hyphal polarity in A. nidulans.

Functional characterization of Aspergillus nidulans homologues of Saccharomyces cerevisiae Spa2 and Bud6.

The importance of polarized growth for fungi has elicited significant effort directed at better understanding underlying mechanisms of polarization, with a focus on yeast systems. At sites of tip growth, multiple protein complexes assemble and coordinate to ensure that incoming building material reaches the appropriate destination sites, and polarized growth is maintained. One of these complexes is the polarisome that consists of Spa2, Bud6, Pea2, and Bni1 in Saccharomyces cerevisiae. Filamentous hyphae differ in their development and life style from yeasts and likely regulate polarized growth in a different way. This is expected to reflect on the composition and presence of protein complexes that assemble at the hyphal tip. In this study we searched for polarisome homologues in the model filamentous fungus Aspergillus nidulans and characterized the S. cerevisiae Spa2 and Bud6 homologues, SpaA and BudA. Compared to the S. cerevisiae Spa2, SpaA lacks domain II but has three additional domains that are conserved within filamentous fungi. Gene replacement strains and localization studies show that SpaA functions exclusively at the hyphal tip, while BudA functions at sites of septum formation and possibly at hyphal tips. We show that SpaA is not required for the assembly or maintenance of the Spitzenkörper. We propose that the core function of the polarisome in polarized growth is maintained but with different contributions of polarisome components to the process.

The Spitzenkörper: a molecular perspective.

The Spitzenkörper is a dynamic structure present at the tips of hyphal cells with a single highly polarized growth site. It is closely connected with cell morphogenesis and polar growth, and is only present at actively growing sites. Morphogenesis of such highly polarized cells is complex, and requires the coordinated action of multiple protein complexes. We discuss the relevance of these complexes for the structure and function of the Spitzenkörper.

A mutation in the Neurospora crassa actin gene results in multiple defects in tip growth and branching.

Actin has a pivotal function in hyphal morphogenesis in filamentous fungi, but it is not certain whether its function is equivalent to that of a morphogen, or if it is simply part of a mechanism that executes orders given by another regulatory entity. To address this question we selected for cytochalasin A resistance and isolated act1, the first actin mutant in Neurospora crassa. This mutant branches apically and shows an altered distribution of actin at the tip. Based on the properties of this mutant, we propose a model of tip growth and branching in which actin effects tip growth by regulating the rate of vesicle flow from proximal to distal regions of a hypha, thereby controlling the tip-high gradient of cytoplasmic calcium. The actin-controlled calcium gradient at the tip is necessary for maintenance of tip growth as well as the dominance of one polarized site at the hyphal tip. The phenotype of act1 indicates that actin controls the balance between lateral and apical branching.